Lysyl oxidase is a copper dependent amine oxidase that catalyses the oxidation of amines, including but not limited to primary amines and, in particular, the amine side chain of lysine. Lysine oxidation catalysed by lysyl oxidase has been observed in the oligopeptide and polypeptide chains of collagen and tropoelastin. Lysyl oxidase activity has been observed with other amine containing substrates, such as oligopeptides where the efficiency of catalyzed oxidation is dependent on adjacent sequences (Kagan et al., 1995a) such as vicinal dicarboxylic amino acid residues (Nagan and Kagan, 1994).
With varying efficiencies, lysyl oxidases can oxidise other substrates, such as butylamine and p-hydroxybenzylamine, for example, to form butyraldehyde and p-hydroxybenzylaldehyde respectively. Non-peptide reactivity is also displayed with, for example, semi-carbazide(s) and oxidation of tyramine (Palcic et al., 1995). Lysyl oxidase activity has also been observed with other amine containing molecules including inhibitors and unnatural substrates such as trans-2-phenylcyclopropylamine (Shah et al., 1993).
The majority of the work done to date in characterising lysyl oxidase has been with respect to non-human mammalian lysyl oxidases. The level of amino acid homology between lysyl oxidases from different species is of the order of 90%.
"Lysyl oxidase-like proteins" have also been identified by the analysis of nucleotide and predicted amino acid sequence alignments of DNA and protein molecules which are expressed in a particular mammalian species including humans. The level of homology between these proteins and lysyl oxidases is of the order of 75%, which is a highly significant degree of homology. Evidence indicates that these molecules may function as enzymes in the extracelluar space as members of a lysyl oxidase family of molecules, and in the cell to provide lysyl oxidase activities (Kim, Y. et al. 1995).
The lysyl oxidase gene encodes a single polypeptide species and there has been no observation of lysyl oxidase mRNA splice variants or isoforms of the lysyl oxidase polypeptide in vivo (Boyd et al., 1995).
In the production of intra- and inter-molecular crosslinked molecules, lysine oxidation in tropoelastin is a necessary step in the formation of allysine, desmosine and isodesmosine condensation products.
Crosslinked molecules, including elastin and collagen, are significant components of fully functional connective tissue. In this regard, deficiencies in lysyl oxidase such as that found in lathyrism, lead to marked phenotypic changes that can compromise the viability of an individual.
Lysyl oxidase exists in at least two forms: an intracellular protein and the more thoroughly characterised extracellular form. In the intracellular form its roles include a ras recission function, and the encoding gene has been classified in this context as a ras-recission gene or rrg (Contente et al., 1990; Kenyon et al., 1991). In its capacity as rrg, its expression is altered in an incompletely catalogued manner, to apparently reduce the oncogenic phenotype of cells expressing aberrant ras. Lysyl oxidase levels also change during differentiation and development (Dimaculangan et al., 1994) and in response to growth factors (Green et al, 1995). Lysyl oxidase is also a secreted protein, available in the extracellular matrix of some connective tissues in very low concentrations.
Difficulties in obtaining sufficient quantities of enzyme for biochemical analysis have impeded detailed exploration of its properties. Yields of purified naturally occurring lysyl oxidase available from typical purification procedures have been limited to 2-4 mg starting with 0.5 to 1 kg of cleaned bovine aortae (Kagan and Cai, 1995).
Extracellular lysyl oxidase is typically made as a larger protein, which includes a collection of amino acid residues at the amino terminus of the protein. This form, termed preprolysyl oxidase is secreted from the cell in a process contemporaneous with cleavage of the amino-terminal region to generate prolysyl oxidase which in turn is cleaved outside the cell to generate the mature form of the protein.
The extracellular (and possibly intracellular) lysyl oxidase enzyme additionally contains copper and at least one organic cofactor which is postulated to be a quinone-like component. The organic cofactor has variously been considered to be covalently or non-covalently bound to the enzyme (Williamson et al., 1986; Kagan and Trackman, 1991). Examples of organic cofactors, which have been found to be associated with oxidases, include quinones such as P.Q.Q., topa quinone (2,4,5 trihydroxyphenylalanine quinone) and tryptophan tryptophylquinone (Tanizawa, 1995). The organic cofactor for lysyl oxidase is now known to be a quinone. It is possible that the cofactor can be supplied, generated by interaction of a quinone derivative with the depleted apo-form of the enzyme, and/or generated by protein oxidation such as that mediated by the participation of copper. There is at least one atom of copper tightly bound per one molecule of functional enzyme. However it was experimentally demonstrated (Gacheru et al., 1990) that when copper is removed by a chelating agent the inactive apoenzyme can be restored to its former level of activity by the addition of copper.
Posttranslational modification(s) such as glycosylation have not been described for the mature form of lysyl oxidase, although there is evidence for glycosylation of the amino terminal region destined for removal during maturation of the precursor prolysyl oxidase (Cronshaw et al., 1995). As other forms of modification exist in vertebrate cells, and include participation by the cytosol, Golgi and secretion machinery, followed by extracellular processing, uncertainty has surrounded the question of whether a functional lysyl oxidase could be made in host cells other than verterbrate systems.
Isolation of endogenous lysyl oxidase from, for example, mammalian tissue typically uses chemical agents which interfere with protein association, such as urea. Modest amounts of lysyl oxidase can be recovered in this way from skin (Shackleton and Hulmes, 1990). The purified material is typically maintained in solutions containing chemical agent(s), in part to minimise protein aggregation and loss of catalytic function. When prepared in this way, the enzyme displays sluggish activity, which has led to the assertion that the enzyme (or a macromolecular complex) (Cronshaw et al.,1993) is altered during the relatively harsh extraction procedure (Shackleton and Hulmes, 1990).
To circumvent difficulties associated with the use of the purified naturally occurring enzyme, recombinant production is a logical alternative. However despite the fact that inferred lysyl oxidase sequences including human (Mariani et al., 1992; Svinarich et al., 1992; Kenyon et al., 1993), rat (Trackman et al., 1990) and mouse (Contente et al., 1993) have been available for several years there have been no reports in the literature of production of recombinant functional lysyl oxidase using non-mammalian hosts for other than modest levels of expression and no functional expression of the human sequence. Kagan et al. (1995b), have reported the production of modest levels of porcine lysyl oxidase from cDNA in mammalian cells indicating that bacterial production of functional enzyme was problematic. One of the impediments to the production of recombinant lysyl oxidase has been the discrepancies appearing in some of the available sequence information (e.g. Trackman et al., 1990; Hamalainen et al., 1991 vs Mariani et al., 1992).